The present invention concerns new bioresorbable polysaccharide sponges, a method for their preparation and uses thereof for the cultivation of mammalian cells in vitro, as well as the use thereof as matrices, supports or scaffolds for implantation into a patient to replace damaged or removed tissue, the polysaccharide sponge implant serving as a substrate, matrix or scaffold for surrounding host tissue to invade it, proliferate thereon and eventually form an active part of the tissue or organ in which the implant was made, or the implant serving as an initial substrate for vascularization from the surrounding host tissue, and once vascularized, cells of choice grown in vitro or obtained from the host may be injected into the vascularized implant to enable a rapid acclimatization and proliferation of the cells which will subsequently form an active replacement for the organ of tissue that was damaged or removed. The polysaccharide sponge can also serve as a substrate, matrix or scaffold for the transplantation of cells initially grown thereon in vitro into a patient to replace damaged, removed or non-functioning tissue.
Porous, absorbable matrices fabricated from natural and synthetic polymers (see, for example, Yannas 1990; Natsumi et al., 1993; Grande, 1989; Vacanti, 1990; Mikos et al., 1993a; Mikos et al., 1993b Mikos et al. 1993c; and Langer and Vacanti, 1993), currently in use or under investigation as implants to facilitate regeneration of tissue in defects caused by disease, trauma or reconstructive surgical procedures. These matrices have been used alone or seeded with cells for the purpose of cell and tissue transplantation (Langer and Vacanti, 1993). Cell transplantation can provide an alternative treatment to whole organ transplantation for failing or malfunctioning organs such as liver and pancreas. As many isolated cell populations can be expanded in vitro using cell culture techniques, only a very small number of donor cells are needed to prepare a suitable implant. Consequently, when such cells are taken from a living donor, the living donor need not sacrifice an entire organ. Furthermore, for the purpose of gene therapy, gene transfer vectors can be introduced into various types of cells, such as, for example, hepatocytes, fibroblasts, keratinocytes, endothelial cells, and myoblasts, which are then transplanted back to the host for the production and local release of proteins and other therapeutic drugs or agents.
Another application of porous matrices has been as scaffolds to investigate the behavior of cells in a three-dimensional framework in vitro (Jain et al., 1990; Doane and Birk, 1991). In some applications, these porous matrices are designed to serve as analogues of the extracellular matrix in order to provide a suitable substrate for cell attachment to enable certain anchor-dependent processes such as migration, mitosis, and matrix synthesis (Folkman and Moscona, 1978). In this regard, it is considered that such analogues of the extracellular matrix may be able to modulate cell behavior in a similar fashion to the way in which the native extracellular matrix does so (see Madri and Basson, 1992), it being believed that the chemistry of these analogues, as well as their pore characteristics such as percentage porosity, pore size and orientation, may influence the density and distribution of the cells within the matrix and thereby affect the regeneration process when these analogues are used in transplantations.
Bioresorbable sponges can also provide a temporary scaffolding for transplanted cells, and thereby allow the cells to secrete extracellular matrix of their own to enable, in the long term, a complete and natural tissue replacement. The macromolecular structure of these sponges is selected so that they are completely degradable and are eliminated, once they have achieved their function of providing the initial artificial support for the newly transplanted cells. For these sponges to be useful in cell transplantations, they must be highly porous with large surface/volume ratios to accommodate a large number of cells, they must be biocompatible, i.e., non-toxic to the cells that they carry and to the host tissue into which they are transplanted, they must be capable of promoting cell adhesion and allowing the retention of the differentiated function of attached cells.
However, in most of the porous matrices described to date, the ones that have been successfully prepared and used in implants or transplants have been limited to those which carry a very thin layer of cells, being principally those which serve as skin substitutes or replacements (see, for example, Yannas, 1990). In view of this limited application, the matrices developed are ones in which the porosity and pore size thereof has been of the type that has been nearly sufficient to allow the dispersion of the thin layer of cells within the matrix. However, when such matrices are to be used with cells such as, for example, hepatocytes, which grow in aggregates of cells and with a thickness greater than the thickness which these earlier matrices are designed to support, a serious problem arises as regards the adequate diffusion of oxygen and nutrients to the inner cells within the matrix, with the result that these inner or lower layers of cells usually die. Thus, these earlier matrices may be useful for preparing skin equivalents, but are much less useful for preparing functional organ equivalents made up of multilayer cell aggregates, both in vitro and with subsequent transplantation use in vivo.
Most of the porous matrices developed to date, as noted above, are based on natural polymers such as collagen, or synthetic polymers from the lactic/glycolic acid family. The collagen-based matrices have several disadvantages, including: they degrade at relatively rapid rate; many disappearing as early as 4 weeks postimplantation (see Olde Damink et al., 1995; Ben-Yishay et al., 1995). Although the rate of degradation of the collagen matrix may be reduced by cross-linking with glutaraldehyde, the resulting cross-linked matrices, however, exhibited immunogenicity, calcification, and fibrous scarring when implanted for long periods (see Timple et al., 1980). Furthermore, collagen matrices are also not suitable for prolonged in vitro cultivation of cells due to a significant contraction of the collagen scaffold, which occurs after approximately one week of incubation, rendering this collagen scaffold less amenable to surgical handling when intended for use as a transplantation matrix (Ben-Yishay, 1995).
Other synthetic biodegradable foams based on poly(D, L-Lactic-co-glycolic acid) have been developed as scaffolds for tissue engineering, as noted above, but because these polymers are hydrophobic, when a cell suspension or culture media is placed on these foams or injected into their interior, the majority of their pores remain empty, resulting in he underutilization of the volume of these foams. In addition, studies have also shown that the degradation of these biodegradable foams results in the signifcant accumulation of acid products which significantly decreases the internal pH within the foam to less than pH 3.0 (see Park Lu and Crotts, 1995), which acidity is very harmful to the growing cells.
Alginates have also been used previously for the purpose of cell transplantation. Aiginates are natural polysaccharide polymers, the word xe2x80x9calginatexe2x80x9d actually referring to a family of polyanionic polysaccharide copolymers derived from brown sea algae and comprising 1,4-Linked xcex2-D-mannuronic (M) and xcex1-L-gluronic acid (G) residues in varying proportions. Alginate is soluble in aqueous solutions, at room temperature, and is capable of forming stable gels, particularly in the presence of certain divalent cations such as calcium, barium, and strontium. The unique properties of alginate, together with its biocompatibilty (see Sennerby et al., 1987 and Cohen et al., 1991), its relatively low cost and wide availability have made alginate an important polymer in medicinal and pharmaceutical applications. For example, it has been used in wound dressings and dental impression materials. Further, alginate has also been approved by various regulatory authorities as acceptable for use as a wound dressing and as food additives in humans. Moreover, pharmaceutical grade alginates, which comply with all the quality and safety requirements of the European and United States of America (USA) pharmacological regulatory authorities, are readily available from several commercial manufacturers. Thus, while alginate has been used for cell transplantation, these previous efforts have generally focused on systems in which a semipermeable membrane was developed as deemed necessary for the protection of cells from the host immune system (see, for example, King et al., 1987 and Sun et al., 1987). These semipermeable membranes were prepared by dropping a mixture of the cells suspended within an alginate solution into a second solution contain calcium chloride. This yielded alginate beads or microcapsules which encapsulated the cells carried thereby. Microcapsules are usually also subjected to a second step in which a semipermeable membrane is formed around the alginate bead by the adsorption of a polycation, such as polylysine, onto the surface of the beads. This coating, however, greatly reduces the microcapsule permeability towards nutrients, which leads to the death of the encapsulated cell.
In view of the above drawbacks of the prior art, it is the aim of the present invention to provide a polysaccharide polymer scaffold made from any suitable polysaccharide polymer, such as, for example, alginates, gellan, gellan gum, xanthan chitosan, agar, carrageenan (polyanionic polysaccharide polymers), or chitosan (polycationic polysaccharide polymers), which provides adequate sites for the attachment and growth of a sufficient cell mass to survive and function not only in vitro but also in vivo, and which polysaccharide polymer scaffold, substrate or matrix, also does not limit the survival and growth of only those cells adjacent to the matrix surface as the cells increase in number within the matrix, but rather also serves to support thick layers of cells, such as cell aggregates, and is capable of maintaining the cells in an active functional state before and after implantation/transplantation into a host tissue, at which time this polysaccharide matrix will also be amenable to vascularization from the surrounding tissue (antgienesis).
It is another aim of the invention to provide polysaccharide matrices which are biodegradable but which de grade only slowly in vivo and thereby permit the cells carried thereby to become established and to form their own tissue matrix at the site of transplant to the point where they no longer require the polysaccharide matrix; or when the matrix is used alone as an implant, it is to be stable for sufficient time for the surrounding tissue to invade it and proliferate thereon to the point where the invasive cells have become established and have formed their own tissue matrix, thereby replacing the originally deficient tissue; or when the matrix is used alone as a first stage of an implant, it is to be stable for sufficient time to allow for vascularization from the surrounding tissue into the implant to occur by invasion thereof by blood vessels, and to allow for the second stage in which cells of choice can be injected into the vascularized matrix and subsequently proliferate thereon to the point where these cells have become established, have formed their own tissue matrix, and thereby have replaced, at least functionally, the originally deficient or damaged tissue.
Yet another aim of the present invention is to provide such polysaccharide sponges of a highly porous nature that may be readily invaded by blood vessels and/or cells and subsequently which may adequately support the growth, proliferation and biological function of such implanted or transplanted cells, both in vitro and afterwards in vivo when used in implants or transplants, such polysaccharide sponges being of a morphology such that their internal volume is optimally utilized, and further providing such sponges which do not require any form of external coating or the like for the purposes of implantation or transplantation, and hence the sponges will not be simply vehicles for the encapsulation of cells but rather would serve as a matrix or scaffold for the cells which they carry and permit free transport of oxygen and nutrients into the cells and in vivo provide for vascularization of the cells for the purposes of nutrient supply and subsequent tissue regeneration from these transplanted cells. Hence, the polysaccharide sponges of the present invention are designed not to serve as merely encapsulation devices for cells but rather as effective matrices, supports or scaffolds for optimal use in implantations and transplantations for tissue repair, as noted above.
A still further object of the present invention is to provide a method for the production of the polysaccharide sponges of the invention.
Other aims of the invention include providing polysaccharide sponges for use in vitro mammalian cell culture, in implantations and in transplantations for repair of damaged or diseased tissue, as well as the use of such polysaccharide sponges for these purposes of in vitro cell culture, implantations and transplantations.
Other objects and aspects of the invention will be readily apparent from the following description of the invention or will arise clearly therefrom.
The present invention is based on a new method for the preparation of three-dimensional, porous, biodegradable sponges and the sponges produced thereby, which sponges are made from any suitable polysaccharide such as, for example, polyanionic polysaccharide polymers, which include alginates, gellan, gellan gum, xanthan chitosan, agar, carrageenan, and polycationic polysaccharide polymers which include chitosan. The polysaccharide sponges of the invention have many possible applications, in particular, medical applications such as, for example, they may be used as a cell matrix, substrate or scaffold to grow various mammalian cells in vitro under conditions that will provide for the obtention of such mammalian cells in vitro that are in an active stage of cell proliferation or even at stages of differentiation with related biological activity of the cells at these stages. Such cellular growth, activation and/or differentiation and/or proliferation is fully dependent on the nature of the substrate, matrix or scaffold on or in which they are grown, and in this regard the new alginate sponges of the invention have been shown to be particularly advantageous for the growth of mammalian cells such as, for example, fibroblasts and hepatocytes. Moreover, mammalian cells grown on or within the polysaccharide sponges in accordance with the present invention may be used in auto and allo transplants for the purposes of, for example, replacing damaged organs or tissues, such as for example skin, liver and many others. Likewise, the polysaccharide sponges of the invention also are particularly useful as implants being inserted into a patient to replace tissue that has been damaged, or removed and which implants are intended to fill the space left by the damaged or removed tissue and to allow for the surrounding tissue to invade the implant and ultimately to fill the implant with the cellular material to restore the originally damaged or removed tissue. Such implants may also be used in a two-stage procedure, in which, in the first stage the implant is inserted into a patient to replace tissue or an organ that has been completely or partially removed. The implant is then invaded by blood vessels from the surrounding tissue, to provide vascularization of the implant, this taking place shortly after implantation. Once the implant has been vascularized, the second stage is performed by injecting into the implant cells of choice which are intended to replace the original tissue/organ. These cells have been previously cultured in vitro or have been obtained fresh from the patient or a suitable donor. Once injected the cells are capable of a rapid acclimatization due to the preformed vascular network in the implant from the first stage. As a result, the injected cells can rapidly proliferate and fill the implant and subsequently differentiate to various stages and ultimately provide an active replacement for the originally damaged or removed tissue/organ. The nature of the polysaccharide sponges of the invention are particularly useful for the aforesaid transplantation or implantation applications in that the polysaccharides of choice are those having a very low immunogenicity, a stability for relatively long periods of time, and because the sponges are biodegradable they will eventually, after a relatively long period of time, be broken down within the body without any deleterious side effects.
The porosity and sponge morphology of the polysaccharide sponges of the invention are dependent on various formulation and processing parameters which may be varied in She process of the invention, and hence it is possible to produce a wide variety of sponges of macroporous nature suitable for cell culture and vascularization. The various sponges have good mechanical properties and hence are suitable, as noted above, to support the growth and proliferation of a wide variety of mammalian cells, such as, for example, fibroblasts and hepatocytes, and thereby the sponges of the invention seeded with such cells can provide at least a temporary support for such cells when transplanted to replace, for example, skin (dermis fibroblasts) or liver (hepatocytes) tissue. This temporary support will be for the period until which the cell transplanted sponge is biodegraded with the patient, at which time it would be expected that by way of the transplant, the originally damaged or diseased tissue would have been able to repair itself.
The new process of the invention is based on a three-step procedure involving a gelation step in which a polysaccharide solution is gelated in the presence of a cross-linking agent, followed by a freezing step, and finally a drying step, by lyophilization, to yield a porous sponge. By altering the conditions at each stage, in particular the concentration of the polysaccharide, the presence or absence of a cross-linking agent and the concentration thereof, the shape of the vessel in which the gelation step is carried out, and the rapidity of the freezing step, it is thereby possible to obtain a very broad range of polysaccharide sponges of various shapes, having various pore sizes and distribution and hence also varying mechanical properties.
The following meanings of various terms will be used herein throughout:
Pore sizexe2x80x94The pore size of a pore within a polysaccharide sponge is determined by using the equation
d={square root over (lxc3x97h)},
wherein l and h are the average length and width of the pores, respectively, as determined by microscopic analysis of the various sponges (see Example 2).
Pore wall thicknessxe2x80x94This parameter characterizes the distance between the pores within a sponge and hence is indicative of the microstructure of the sponges and is determined also by measurement at the microscopic level of the various sponges (see Example 2).
E-modulus of elasticityxe2x80x94This is a measure of the relative rigidity of the polysaccharide sponges and is determined in units of kPa when subjecting sponges to compression and monitoring the rate of their deformation. The higher the E-modulus of elasticity, the higher is the relative rigidity of the sponge.
Polysaccharide solutionsxe2x80x94This is taken to mean two kinds of solutions, the first being the original solution of the polysaccharide in water, prepared by dissolving under conditions of homogenization, a commercially available form of the polysaccharide in water, usually yielding a solution of the salt of the polysaccharide, for example, a sodium alginate solution. This initial solution is then subjected to gelation as the first step in the sponge preparation process of the invention. The solution subjected to gelation is called the final polysaccharide solution, and in many cases is a further diluted form of the initial polysaccharide solution. Hence, when concentrations of polysaccharide are indicated herein throughout, they usually refer to the concentration of the polysaccharide in the final solution that was subjected to the gelation step in the first part of the process from which the polysaccharide sponge is obtained. In the examples herein below there is exemplified a variety of sponges made from but one of the polysaccharides of choice, namely, various alginates. Hence, in accordance with the above-mentioned, there will be used xe2x80x9coriginal alginate solutionxe2x80x9d or xe2x80x9cinitial alginate solutionxe2x80x9d to indicate the aqueous alginate solution fast form by dissolving an alginate powder in water, and xe2x80x9cfinal alginate solutionxe2x80x9d to indicate the dissolved alginate solution subjected to gelation and subsequent freezing and drying.
Implantationxe2x80x94This term is usually meant to imply the insertion of a polysaccharide sponge of the invention into a patient, whereby the implant serves to replace, fully or partially, tissue that has been damaged or removed with the implant serving, as a matrix, substrate or scaffold on which surrounding tissue which may invade the implant and may grow so that ultimately, following sufficient growth of such tissue within the sponge and with the biodegradation of the sponge over time, the injured or removed tissue will be effectively replaced. Implantation in this sense also means the above-noted two-stage procedure in which, in the first stage, the implant is placed into the patient and becomes vascularized by invasion of blood vessels from surrounding tissue, a process which usually occurs rapidly following implantation. Once vascularized, the implant is then accessible to the second stage being the the injection thereinto of cells of choice either grown previously in vitro or obtained from the patient or a suitable donor. Such cells are capable of a rapid acclimatization because of the pre-formed vascular network within the implant, and hence are also capable of a rapid proliferation and subsequent functional differentiation to provide a replacement for the damaged or removed tissue. In fact, when it is necessary to fully replace a tissue/organ, for example, skin or liver segments or portions that have been removed, then there is a need to apply the above two-stage procedure. In this way functional cells, for example, fibroblasts or hepatocytes, will be infected into the implant which has already been vascularized. Another aspect of implantation is also take to mean the use of a polysaccharide sponge as a vehicle to transport therapeutic drugs to a certain site in a patient, usually by, way of cells carried by the polysaccharide sponge which are capable of secreting a desired therapeutic protein, hormone or the like, or which secrete various regulatory proteins which in turn can direct the expression of such required therapeutic drugs endogenously within the tissue in which the implant has been inserted. In this aspect there is also included the introduction into the polysaccharide sponge of encapsulated therapeutic agents, for example, growth factors, angiogenic factors, and the like, which are advantageous to encourage a more rapid growth of the cells within the implant, or a more rapid vascularization of the implant. Such factors are usually too small to be effectively retained within the sponge and hence are introduced in the form of slow-release or controlled-release microcapsules into the sponge to provide for their effectivity.
Transplantationxe2x80x94Transplantation may be of two kinds, i.e., allo or auto transplantation, and in both cases, the cells to be transplanted will first be grown, in vitro on or within the alginate sponge until they reach a desired state of cell activation, proliferation of differentiation as required, at which time the alginate sponge with such seeded cells will be transplanted into a patient at the desired site for the purposes of organ or tissue repair, or replacement. As noted above, the transplantation can also include, besides the cells, microcapsules containing therapeutic agents for the cells, vascularization or for the host.
Accordingly, the present invention provides a polysaccharide sponge characterized by having: (i) an average pore size in the range between about 10 xcexcm to about 300 xcexcm; (i) an average distance between the pores being the wall thickness of the pores in the range between about 5 xcexcm to about 270 xcexcm and (iii) an E-modulus of elasticity being a measure of the rigidity of the sponge in the range of about 50 kPa to about 500 kPa.
An embodiment of the sponge of the invention is a sponge which comprises a polysaccharide selected from the group comprising the polyanionic polysaccharides: alginates, gellan, gellan gum, xanthan chitosan, agar, carrageenan and the polycationic polysaccharide: chitosan.
Another embodiment of the polysaccharide sponge of the invention is a sponge which comprises an alginate selected from the group of alginates characterised by having: (i) a mannuaronic acid (M) residue content in the range of between about 25% and about 65% of total residues: (ii) a guluronic acid (G) residue content in the range of between about 35% and about 75% of total residues; (iii) a M/G ratio of about 1/3 and about 1.86/1; and (iv) a viscosity of the final alginate solution having 1% w/v alginate, from which the sponge is obtained in the range between about 50 cP to about 800 cP.
Preferred polysaccharide sponges of the invention include sponges comprising an alginate derived from brown sea algae selected from the group consisting of alginate Protanal(trademark) LF 120 (LF 120) derived from Laminaria hyperborea, alginate Protanal(trademark) LF 20/60 (LF 20/60) derived from Laminaria hyperborea, alginate MVG(trademark) (MVG) derived from Laminaria hyperborea, alginate Pronatal(trademark) HF 120 (HF 120) derived from Laminaria hyperborea, alginate Pronatal(trademark) SF 120 (SF 120) derived from Laminaria hyperborea, alginate Pronatal(trademark) SF 120 RB (SF 120 RB) derived from; Laminaria hyperborea, alginate Pronatal(trademark) LF 200 RB (LF 200 RB) derived from Laminaria hyperborea, alginate Manugel(trademark) DMB (DMB) derived from Laminaria hyperborea, Keltone(trademark) HVCR (HVCR) derived from Macrocystis pyrifera, and Keltone(trademark) LV (LV) derived from Macrocystis pyrifera. 
The above alginate sponges of the invention preferably are formulated wherein the alginate is used in the form of a sodium alginate solution having a concentration of alginate between about 1% to about 3% w/v to provide an alginate concentration between about 0.1% to about 2% w/v in the final solution from which the sponge is obtained.
In accordance with yet another embodiment of the invention, the polysaccharide sponges may also comprise a cross-linking agent selected from the group consisting of the salts of calcium, copper. aluminum, magnesium, strontium, barium, tin, zinc, chromium, organic cations, poly(amino acids), poly(ethyleneimine), poly(vinylamine), poly(allylamine), and polysaccharides.
The most preferred cross-linking agents for use in the preparation of the sponges of the invention are selected from the group consisting of calcium chloride (CaCl2), strontium chloride (SrCl2) and calcium gluconate (Ca-Gl).
Preferably, the cross-linker is used in the form of a cross-linker solution having a concentration of cross-linker sufficient to provide a cross-linker concentration between about 0.1% to about 0.3% w/v in the final solution from which the sponge is obtained.
The preferred polysaccharide sponges of the invention are those which are prepared from a polysaccharide solution with or without the addition of a cross-linker. Embodiments of these preferred sponges of the invention include an alginate sponge prepared from an alginate solution with or without the addition of a cross-linker and wherein said final alginate solution with or without cross-linker from which said sponge is obtained is selected from the group of final solutions, having concentrations of alginate or alginate and cross-linker, consisting of: (i) LF 120 alginate 1% w/v without cross-linker; (ii) LF 120 alginate 1% w/v and Ca-Gl 0.1% w/v; (iii) LF 120 alginate 1% w/v and Ca-Gl 0.2% w/v; (iv) LF 120 alginate 1% w/v and SrCl2 0.15% w/v; (v) LF 120 alginate 1% w/v and CaCl2 0.1% w/v; (vi) LF 120 alginate 0.5% w/v and Ca-Gl 0.2% w/v; (vii) LF 20/60 alginate 1% w/v and Ca-Gl 0.2% w/v; (viii) HVCR alginate 0.5% w/v and Ca-Gl 0.2% w/v; and (ix) HVCR alginate 1% w/v and Ca-Gl 0.2% w/v.
Other such embodiments include sponges obtained from a final solution of LF 120 alginate 1% w/v and Ca-Gl cross-linker 0.2% w/v; and a sponge obtained from a final solution of HVCR alginate 1% w/v and Ca-Gl cross-linker 0.2% w/v.
The present invention also provides a process for producing a polysaccharide sponge of the invention comprising:
(a) providing a polysaccharide solution containing about 1% to about 3% w/v polysaccharide in water;
(b) diluting said polysaccharide solution with additional water when desired to obtain a final solution having about 0.5% to about 2% w/v polysaccharide, and subjecting said solution of (a) to gelation, to obtain a polysaccharide gel;
(c) freezing the gel of (b); and
(d) drying the frozen gel of (c) to obtain a polysaccharide sponge.
An embodiment of the above process of the invention is a process further comprising the addition of a cross-linker to said polysaccharide solution of (a) during the step of elation (b), said cross-linker being added in an amount to provide a concentration of cross-linker in the final solution being subjected to gelation of between about 0.1% to about 0.3% w/v.
In a preferred embodiment of the gelation step (b) of the process of the invention, the gelation is carried out by intensive stirring of the polysaccharide solution in a homogenizer at about 31800 RPM for about 3 minutes, and wherein when a cross-linker is added to the solution, said cross-linker is added very slowly during said intensive stirring of the alginate solution.
In a preferred embodiment of the process of the invention, there is provided a process wherein the polysaccharide is an alginate selected from the group consisting of an alginate derived from brown sea algae selected from the group consisting of alginate Protanal(trademark) LF 120 (LF 120) derived from Laminaria hyperborea, alginate Protanal(trademark) LF 20/60 (LF 20/60) derived from Laminaria hyperborea alginate MVG(trademark) (MVG) derived from Laminaria hyperborea, alginate Pronatal(trademark) HF 120 (HF 120) derived from Laminaria hyperborea, alginate Pronatal(trademark) SF 120 (SF 120 ) derived from Laminaria hyperborea, alginate Pronatal(trademark) SF 120 RB (SF 120 RB) derived from Laminaria hyperborea, alginate Pronatal(trademark) LF 200 RB (LF 200 RB) derived from Laminaria hyperborea, alginate Manugel(trademark) DMB (DMB) derived from Laminaria hyperborea, Keltone(trademark) HVCR (HVCR) derived from Macrocystis pyrifera, and Keltone(trademark) LV (LV) derived from Macrocystis pyrifera. 
In the above preferred embodiment of the process of the invention, when the polysaccharide is alginate, the preferred final solutions containing alginates with or without cross-linker that are subjected to the gelation step (b) are the following: (i) LF 120 alginate 1% w/v without cross-Linker; (ii) LF 120 alginate 1% w/v and Ca-Gl 0.1% w/v; (iii) LF 120 alginate 1% w/v and Ca-Gl 0.2% w/v; (iv) LF 120 alginate 1% w/v and SrCl2 0.15% w/v; (v) LF 120 alginate 1% w/v and CaCl2 0.1% w/v; (vi) LF 120 alginate 0.5% w/v and Ca-Gl 0.2% w/v; (vii) LF 20/60 alginate 1% w/v and Ca-Gl 0.2% w/v; (viii) HVCR alginate 0.5% w/v and Ca-Gl 0.2% w/v; and (ix) HVCR alginate 1% w/v and Ca-Gl 0.2% w/v.
The freezing step (c) of the process of the invention may be by rapid freezing in a liquid nitrogen bath at about xe2x88x9280xc2x0 C. for about 15 minutes, or by slow freezing in a freezer at about xe2x88x9218xc2x0 C. for about 8 to 24 hours. The most preferred means of freezing is by rapid freezing in a liquid nitrogen bath as noted above.
The drying step (d) is preferably by way of lyophilization under conditions of about 0.007 mmHg pressure and at about xe2x88x9260xc2x0 C.
For the purposes of preparing the polysaccharide sponges of the invention with various shapes and sizes, for example, nose shapes, cube shapes, cylindrical shapes and the like (see FIG. 2), it is preferable to carry out the process of the invention by pouring the initial polysaccharide solution into an appropriately shaped vessel having the desired shape and performing the gelation and subsequent steps of the process in this shaped vessel.
The present invention also provides an polysaccharide sponge of the invention, in particular alginate sponges, as noted above for use as a matrix, substrate or scaffold for growing mammalian cells in vitro. Another preferred use of the polysaccharide sponges of the invention is their use as a matrix substrate or scaffold for implantation into a patient to replace or repair tissue that has been removed or damaged, wherein said implanted sponge is a substrate, matrix or scaffold for surrounding tissue to invade it, proliferate thereon and replace the damaged or removed tissue, or wherein said implant is an initial substrate for vascularization by the surrounding host tissue and the vascularized implant then serves as a substrate to receive injected cells of choice from the host or grown in vitro, said injected cells being capable of rapid acclimitization and proliferation on the vascularized sponge to rapidly replace the damaged or removed tissue.
Yet another preferred use of the polysaccharide sponges of the invention is the use as an implanted support for therapeutic drug delivery into a desired tissue, said drug delivery being by way of the action of genetically engineered cells or natural cells carried by said sponge and expressing said therapeutic drugs, said cells expressing said drug or expressing regulatory proteins to direct the production of the drug endogenously in said tissue. In this preferred use, the therapeutic drug expressed by the cells carried on or in the sponge is a therapeutic protein wherein said cells express said protein or express regulatory proteins to direct the production of said protein endogenously in the tissue into which said sponge is implanted.
Other preferred embodiments for the use of the polysaccharide sponges in accordance with the present invention include: the use of the sponges as a matrix, substrate or scaffold for vitro culturing of plant cells and algae; for use as a matrix, substrate or scaffold for the delivery to a tissue or organ of genetically engineered viral vectors, non-viral vectors, polymeric microspheres and liposomes all encoring or containing a therapeutic agent for said tissue or organ; for use as a matrix, substrate or scaffold for in vitro fertilization of mammallian oocytes; for use as a matrix, substrate or scaffold for storage of fertilized mammalian oocytes, or other mammalian cells cultured in vitro; for use as a matrix, substrate or scaffold for the storage of plant cells and algae cultured in vitro; and for use as a matrix, substrate or scaffold for the transplantation of cells grown on or within the sponge in vitro into a tissue of a patient in need of the cells as a result of tissue damage, removal or dysfunction.
Preferred uses of the polysaccharide sponge of the invention as noted above include the use thereof for growing fibroblast cells in vitro; and for growing hepatocyte cells in vitro. In accordance with these preferred uses, the polysaccharide sponges of the invention may be used as transplantation devices to transplant fibroblast cells to replace damaged or removed skin tissue, or for the transplantion of hepatocytes to replace damaged or removed liver tissue.
The present invention thus also provides artificial organ equivalents which serve to provide the essential function of the organ which they are to replace fully or partially or whose function they are designed to augment. The artificial organ equivalents of the invention therefore comprise a polysaccharide sponge of the invention, as noted above, and representative cells of the said organ, the cells having been grown on or within the sponge in vitro to the stage wherein they are fully active and equivalent to the active cells of the organ and thereby the artificial organ is suitable for transplantation or implantation in all the various ways thereof as detailed above, into a patient in need thereof following organ damage, removal or dysfunction. Preferred embodiments of the artificial organ equivalents of the invention being artificial skin comprising a polysaccharide sponge of the invention and dermal fibroblast cells, as well as an artificial liver equivalent comprising a polysaccharide sponge of the invention and hepatocytes.